Analysis Of Resistance In Mycobacterium Tuberculosis And Detection Of Genes Resistant To Rifampicin And Ofloxacin

Objective: To investigate the resistance of Mycobacterium tuberculosis(MTB) to four first-line drugs and two second-line drugs, and analyze the mutation characteristics of rpoB, gyrA and gyrB gene in MTB isolates. Methods:(1) Specimens were collected from 6535 tuberculosis patients who presented to Hunan Chest Hospital from January to December in 2014. Liquid culture was performed for the specimens using BACTEC MGIT 960. Drug resistance of Mycobacterium tuberculosis clinical isolates to streptomycin(SM, S), isoniazid(INH, H) and rifampicin(RFP, R),ethambutol(EMB, E),kanamycin(KM, K) and ofloxacin(OFX, O) were detected through absolute concentration method using H37 Rv as control.(2) Genomic DNA of Mycobacterium tuberculosis clinical isolates resistant to rifampicin were extracted by ultrasonic decomposition method, rpoB gene was amplified and sequenced so that the relationship between the mutation of rpoB gene and phenotype was analyzed.(3) Genomic DNA of Mycobacterium tuberculosis clinical isolates resistant to ofloxacin were extracted and used as template to amplify the genes gyrA and gyrB. Amplified genes of gyrA and gyrB were sequenced respectively by ABI 3700 DNA sequencer, and the relationship between the mutation of gyrA and gyrB gene and phenotype was anaylzed. Rusults:(1) A total of 1140 tuberculosis patients including 826 new cases and 314 previously treated cases were MTB culture-positive; 443(38.85%) cases including 296 new cases and 147 previously treated cases were drug-resistant tuberculosis in 1140 tuberculosis patients. Overall, 270(23.68%) patients are multidrug-resistant(MDR) tuberculosis(defined as disease that was resistant to at least isoniazid and rifampicin).The dominant MDR pattern was that MTB was resistant to rifampicin, isoniazid and streptomycin simultaneously, which accounted for 32.22%(87/270). Rifampicin-resistance tuberculosis was dominant among the mono-resistance patterns, which accounted for 28.51%(325/1140).(2)The major type of rpoB mutations was C531T(Serâ†'Leu)(69.64%), including 29 multidrug-resistant strains(70.73%), followed by the occurrence on the RRDR-526 loci gene mutations, which accounted for 16.07%.(3) In all 1140 Mycobacterium tuberculosis clinical isolates, 140(12.28%) strains were ofloxacin-resistant. Mutations of gyr gene occurred in 25 of 32(accounted for 78.1%) ofloxacin drug-resistant strains. Among gyrA gene mutations, 94 loci was common, which accounted for 60.0%(15/25). Mutations occurred in gyrB gene were not observed. Conclusion:(1)The rate of total resistance and multi-drug resistance of the tuberculosis patients were 38.85% and 3.68% respectively.(2)The two mutation loci on RRDR-531 and RRDR-526 are the major mutation types conferring RFP resistance.(3) Mutations in the gyr gene are associated with Ofloxacin resistance and mainly occurred in gyrA gene.